Use of ligase chain reaction for enhanced detection of Phytophthora infestans

• Published in: Canadian journal of plant pathology Vol. 24; no. 3; pp. 294 - 301
• Main Authors: Tooley, P.W., Carras, M.M., Hatziloukas, E., Scott, D.L.
• Format: Journal Article
• Language: English
• Published: Taylor & Francis Group 01-09-2002
• Subjects: biotin; colorimetric; digoxigenin; molecular biology; paramagnetic
• ISSN: 0706-0661; 1715-2992
• DOI: 10.1080/07060660209507012

Sequences of the internal transcribed spacer region 2 (ITS 2) of ribosomal DNA from 10 different species of Phytophthora were aligned, and regions of dissimilarity were used to construct ligase chain reaction (LCR) primers for detecting the late blight pathogen, Phytophthora infestans. Ligase chain reaction primers allow differentiation of species down to a single base pair difference. The primers have greater specificity than previously described polymerase chain reaction (PCR) primers and allow differentiation of P. infestans, Phytophthora mirabilis, and Phytophthora phaseoli, which contain identical ITS sequences, from many other Phytophthora species. Ligase chain reaction was coupled with PCR to increase sensitivity of P. infestans detection and can serve as an enhancement to existing PCR protocols. By labeling LCR primers with biotin and digoxigenin, the LCR was combined with an ELISA-based detection system to improve sensitivity and ease of application.